CN106591179B - Methyl packing bacterium and its prepare the application on (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt in selective fractionation - Google Patents
Methyl packing bacterium and its prepare the application on (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt in selective fractionation Download PDFInfo
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Abstract
The application on 2 oxygen of (S) α ethyls, 1 pyrrolidine acetic acid salt is prepared the invention discloses methyl packing bacterium and its in stereoselectivity fractionation, after cell fixation being carried out to the methyl packing bacterium of producing enzyme, applied to biological resolution racemic modification (R, S) 2 oxygen of α ethyls, 1 pyrrolidine acetic acid ethyl ester prepares 2 oxygen of high optical voidness (S) α ethyls, 1 pyrrolidine acetic acid ethyl ester, further obtains 2 oxygen of (S) α ethyls, 1 pyrrolidine acetic acid salt by hydrolysis.The present invention realizes that conversion yield reaches as high as more than 50.0%, and stereoselectivity is good, 2 oxygen of (S) α ethyls, 1 pyrrolidine acetic acid ethyl ester enantiomeric excess value e.e.s(%) is not less than 99.5；High catalytic efficiency, the racemic substrate concentration in resolution reaction reach as high as 500g/L, and the reaction time is no more than 15 hours, and number is reused after cell fixation not less than 35 times, is easy to industrialized production, downstream separation is simple, and environmental pollution is small.
Description
Technical field
The present invention relates to biocatalysis technology fields, relate in particular to methyl packing bacterium and its are prepared selectively splitting
(S) application on-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt.
Background technology
Levetiracetam (Levetiracetam) chemical name is (S)-α-ethyl -2- oxygen conjunction -1- acetamide pyrrolidines,
It is that a kind of the novel of acetyl pyrrole alkyl compound resists insane carbuncle drug.Not only therapeutic index is very high, but also characteristics of pharmacokinetics for the medicine
Uniqueness, oral absorption is fast and safety, bioavilability are a kind of wide spectrum anti-epileptics efficient, toxic side effect is small up to 100%
Medicine has high Development volue.Document report, d-isomer (R)-α-ethyl -2- oxygen conjunction -1- acetamides pyrrolidines is for inhibiting
Epileptic attack only has slight or unconspicuous drug action, and Levetiracetam is then a kind of safe and efficient antiepileptic
Object.
The key of Levetiracetam synthesis is to control the optical purity of levo form during the reaction.(S)-α-ethyl -2-
Oxygen -1- pyrrolidine acetic acids ethyl ester can obtain (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids through ester hydrolysis, and the acid of the configuration is to close
Into the crucial chiral intermediate of Levetiracetam, structural formula is as follows：
Single enantiomter can be by preparations such as chemistry, enzyme process or chemo-enzymatic process.Early literatures describe left second and draw
Western smooth preparation process：By racemic levetiracetam intermediate (±)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids and optics
Pure resolution reagent (R)-(+)-α-phenylethylamine or chiral phosphine generation salt, then crystallisation by cooling, be isolated out corresponding 2 it is right
Reflect body.During traditional Chiral Separation there is high energy consumption, cumbersome, yield is relatively low, product purity is relatively low, environment
The problems such as seriously polluted, is unfavorable for pharmacy corporation energy-saving and emission-reduction.
Compared with chemical method, the advantages of living things catalysis is that catalysis reaction typically exhibits high stereoselectivity and region choosing
Selecting property, and its catalytic condition is mild, low energy consumption, efficient.Chinese patent " resistance to tyrosine tomb village Salmonella and its catalysis prepare (S)-
α-ethyl -2- oxygen -1- pyrrolidine acetic acids " (publication number CN 101748087A) discloses kind of a resistance to tyrosine tomb village Salmonella, can be used
It is prepared in (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in chiral living things catalysis, the yield of this method reaches 48.1%, but its
Reaction time is long (12~60h), and reaction substrate concentration is too low (5.2~31.2g/L of racemic substrate concentration), and it is excessive to throw enzyme amount
(5.2~31.2g/L of wet thallus cell), it is difficult to realize industrialized production.Chinese patent application " microorganism catalysis prepare (S)-α-
The method and bacterial strain of ethyl -2- oxygen -1- pyrrolidine acetic acid esters " (publication number CN102994429A) discloses a kind of wax-like gemma bar
Bacterium, using racemic ' alpha '-ethyl -2- oxygen -1- pyrrolidine acetic acids esters as substrate, the ester hydrolase which generates is catalyzed stereoselectivity
(R)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids esters are hydrolyzed so as to obtain the method for S anomeric products, substrate maximum is thrown in the technology
Amount only has 100g/L, and the reaction time is up to 60h, and maximum output 48.9% is more difficult applied to realization industrialized production.
Invention content
Goal of the invention：The object of the present invention is to provide a kind of new methyl packing bacterium；A further object of the invention is to carry
The application on (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt is prepared in stereoselectivity fractionation for aforementioned methyl packing bacterium, with
It is produced suitable for industrial-scale metaplasia；It is a still further object of the present invention to provide it is a kind of split it is efficient, be easy to amplification production
Immobilized cell technique.
Technical solution：For the methyl packing bacterium cxzy-L013 (Methylopila sp.cxzy-L013), the bacterium
Kind is preserved in China typical culture collection center (CCTCC), address：Wuhan, China university, 430072, preservation date：2016
On September 18, deposit number：CCTCC NO.：M2016494.
The methyl packing bacterium cxzy-L013 (Methylopila sp.cxzy-L013) is from Linhai city Du Qiao towns
Through flat-plate bacterial colony feature primary dcreening operation in Huahai's medicine company on-site soil, using one grade fermemtation shaking flask culture one by one, by detecting enzyme activity
Property, compare stereoselectivity ester hydrolase enzyme activity size and obtain.
The feature of the bacterium colony is as follows：Colonial morphology：30 DEG C of 48~72h of culture on LB agar plates, bacterium colony are in regular shape
Circle, neat in edge, 0.5~1mm of diameter, surface elevation, moistening, glossy, milky；Thalline be in short round bar shape, single point
Arrangement is dissipated, size is (0.3~0.4) μ m (1.0~1.2) um；Gram-negative bacteria；Using glucose, glycerine, ethyl alcohol as carbon
Slow-growing on the culture medium in source, growth is very fast during using methanol, methylamine hydrochloride, ammonium formate as carbon source.
The present invention provides prepare (S)-α-second in stereoselectivity fractionation using aforementioned methyl packing bacterium cxzy-L013
Application on base -2- oxygen -1- pyrrolidine acetic acid salt, while also provide the methyl packing bacterium cxzy-L013 stereoselectivities
The method that fractionation prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt, includes the following steps：
(1) bacterium solution of the methyl packing bacterium cxzy-L013 is handled by method for immobilizing cell, obtains immobilized bacterium
Agent；
(2) with (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester for substrate, a certain amount of water and described solid is added in
Surely change microbial inoculum and carry out resolution reaction, obtain (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters；
(3) and then using enzyme hydrolysis or basic hydrolysis (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt is obtained.
The bacterium solution of the methyl packing bacterium cxzy-L013 is through inclined-plane culture, seed liquor by methyl packing bacterium cxzy-L013
The bacteria suspension containing enzyme that 50wt% is no less than containing wet thallus that culture, inoculation fermentation step obtain.Specific acquisition step is as follows：
Inclined-plane culture
Methylopila sp.cxzy-L013 glycerol tubes bacterial strains are crossed in LB slant tubes, 30 DEG C are cultivated 2~3 days；
Seed liquor culture
Inclined-plane thalline is seeded to seed culture medium：30 DEG C are cultivated 2~3 days, obtain seed liquor；The seed culture medium concentration
It forms and is：MgSO4·7H2O1.0g/L, K2HPO41.8g/L, (NH4)2SO41.0g/L, yeast extract powder 5.0g/L, volume fraction
For 75% methanol solution 5.0mL/L (being added in before inoculation), ammonium hydroxide adjusts pH 7.0.
Inoculation fermentation
Seed liquor is seeded to ferment tank：Inoculum concentration be 1~10v/v%, 30 DEG C of fermentation temperature, ammonium hydroxide control pH6.5
~7.0, ventilatory capacity：0.5~1vvm, 100~1000r/min of mechanical agitation, the methanol concentration of fermentation process intermittent injecting 75%
For 5.0mL/L, fermentation duration 3~4 days can put tank when pH goes up not down and collect thalline；OD600 >=40 at this time, thalline are wet
Weight is up to 70~90g/L.
The fermentation medium concentration composition：NaCl 0.5g/L, MgSO4·7H2O3.6g/L, K2HPO41.0g/L
(NH4)2SO41.0g/L, yeast extract powder 6.0g/L, the methanol solution 5.0mL/L (being added in before inoculation) that volume fraction is 75%.
Including but not limited to methanol, methylamine hydrochloride, formic acid can be selected in present invention carbon source of culture medium in inoculated and cultured
Any one of ammonium, preferably methanol.When such as using glucose, glycerine, ethyl alcohol instead as carbon source, thalli growth is very slow.
The bacteria suspension containing enzyme of 50wt% is no less than to obtain, zymotic fluid is obtained after supercentrifuge centrifuges containing enzyme
Wet thallus is stirred evenly wet thallus and water dilution by equal quality ratio, and refrigeration is for use；Or directly by zymotic fluid through micro-filtration membrane concentration extremely
Mass fraction containing wet thallus is about the bacteria suspension of 50wt%, and refrigeration is for use.
The method for immobilizing cell is that the bacterium solution of methyl packing bacterium cxzy-L013 is dissolved in buffer solution, is at least added in
A kind of adsorbent and/or a kind of crosslinking agent, stirring filter acquisition immobilized microbial inoculum；The adsorbent is selected from diatomite, activated carbon
Any one；The crosslinking agent is selected from any one of glutaraldehyde, toluene di-isocyanate(TDI), bis-diazotized benzidine.Utilize this
The enzyme activity rate of recovery for the immobilised enzymes that process for fixation is realized is not less than 90%, and adsorption rate is not less than 95%, and immobilized microbial inoculum can
Number is reused to be not less than 35 times.
Enzyme activity rate of recovery calculation formula after cell or enzyme immobilization is：
A=W0/(W1-W2) * 100%
In formula：The enzyme activity rate of recovery of the A for immobilised enzymes, W1To add in the total activity of resolvase, W2For supernatant after immobilization
Total enzyme activity power, W0Total enzyme activity power for immobilised enzymes.
Adsorption rate calculation formula after cell or enzyme immobilization is：
B=(W1-W2)/W1* 100%
In formula：Adsorption rates of the B for immobilised enzymes, W1To add in the total activity of resolvase, W2Enzyme for supernatant after immobilization
Vigor.
In immobilized cell technique, investment, cross-linking method, absorption method, covalent immobilization are common technological means, packet
It buries method generally to embed cell or enzyme using sodium alginate, carragheen etc., but the immobilization particle mechanical strength after embedding
It is smaller, it is easily broken in whipping process.Absorption method is easy to operate, easily realizes immobilization, and mild condition fixation support is cheap
It is easy to get, and can reuse, but the enzyme or cell quantity adsorbed is limited and binding ability between carrier is weaker, it is easily de-
It falls, vigor is caused to decline and pollutes reaction product.The amino of enzyme, carboxyl, phenolic group, sulfydryl, hydroxyl, imidazole radicals in covalent coupling method
Wait functional groups that can participate in covalent bond, wherein it is most commonly seen with the combined techniques of amino and carboxyl, what usual this method obtained
It is firmly combined with being not susceptible to enzyme obscission between enzyme and carrier in immobilised enzymes, but zymoprotein higher structure is easily more acute
It is destroyed under strong reaction condition.Immobilized cell or enzyme stability prepared by cross-linking method is good, and the up time is long；Penta 2
Aldehyde is most common crosslinking agent, it is can be with the functional group on cell or enzyme by the small-molecule substance with difunctional aldehyde radical
Schiff base reaction occurs for amino, by cell or enzyme crosslinking and then achievees the purpose that stable cell or enzymatic structure, but single use is handed over
The immobilized cell gas porosity of connection method is poor, it is difficult to solve immobilized cell and the effective of reaction solution in catalytic reaction process and divide
From.
As advanced optimizing for the present invention, the method for immobilizing cell is first to add in adsorbent, is uniformly mixed
After add crosslinking agent.Form embedding bead, then combine by cross-linking method first with absorption method, can significantly increase mechanical strength and
Improve its stability.
As the further optimization of the present invention, the crosslinking agent is also added into polyethyleneimine when adding in.Polyethyleneimine
Amine has high adhesion and adsorptivity, and amido can generate hydrogen bond with the hydroxyl reaction in cell or enzyme molecule, be reacted with carboxyl
Ionic bond is generated, generation covalent bond can be also also reacted with carbonic acyl radical；Simultaneously as with polar group (amido) and hydrophobic group
(vinyl) constructs, and can be combined with different substances.
As the optimal case of the present invention, the step of cell fixation, is：It is about 50% by mass fraction containing wet thallus
Bacteria suspension 20g is dissolved in 100mL Ammonium formate buffers (pH 7.0), is uniformly mixed, and adds 0.6g diatomite or activated carbon, adds
Enter 5% polyethyleneimine 3mL to be crosslinked 1 hour, then 25% glutaraldehyde 1mL is added to be crosslinked 1 hour, most fixed afterwards through vacuum filtration
Change microbial inoculum, with originally water washing 2 times and filter, be placed in 4 DEG C it is stored refrigerated.
By the way that glutaraldehyde and polyethyleneimine is used in combination, the aldehyde radical of glutaraldehyde polymerize to be formed with the amino of polyethyleneimine
Fine and close reticular structure wraps up somatic cells；Diatomaceous addition contributes to suction-operated, while increase of immobilised enzymes
Granularity and gas porosity make the particle after immobilization more easily separated.
The immobilized microbial inoculum obtained using above-mentioned method for immobilizing cell splits racemic modification substrate, can make substrate
Concentration greatly increases, and makes it possible industrial mass production.(R, the S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid second
Ester is the toluene solution of 500~900g/L, and preferred concentration is in 700~800g/L.
The condition of resolution reaction is in the step 2：Reaction temperature is 25~37 DEG C, and the carbonic acid of 15~25v/v% is added dropwise
Sodium water solution control reaction process pH 6.5~8.5, reacts 2~15 hours.
Further, (R, the S)-α-mass concentration of ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester in the reaction system is
160~500g/L.The immobilized microbial inoculum is in terms of weight in wet base, a concentration of 5~25g/L of throwing amount in the reaction system.
After resolution reaction, reaction conversion results, enantiomeric excess value e.e. are detected using HPLCs(%) is not less than
99.5, conversion ratio is not less than 49%.
Mixed liquor isolation and purification method after resolution reaction is：By reaction solution directly with supercentrifuge centrifugation or board-like mistake
Filter filters, and retains organic phase after liquid portion layering, and the toluene that 1: 0.3~1.5 are added in water layer carries out 3~5 extractions, closes
And organic phase, 35~60 DEG C steam toluene, obtain concentrate (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters.
As advanced optimizing for the present invention, (R)-α-ethyl -2- oxygen -1- pyrroles in water phase is recycled after step (2)
Alkane acetic acid, through racemization and into obtaining starting material raw material after esterification.
It, can be again by immobilized ester hydrolase enzyme for (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters of acquisition
Method for hydrolysis obtains the salt of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids：Add 1 part of industrial water and 1 respectively into reactor
The concentrate of~2 parts of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters adds in 1/20~1/5 part of ester hydrolase, stirs, control
Temperature processed and pH until ester hydrolysis reaction terminates, filter out immobilized ester hydrolase and obtain (S)-α-ethyl -2- oxygen -1- pyrrolidines
The solid crude product of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt is made in the salting liquid of acetic acid, then concentrated crystallization.
The ester hydrolase selects existing ester hydrolase well known to those skilled in the art, is preferably applied to stablize hydrolysis
(S) enzyme of-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters, and reacted under the optimum temperature of the enzyme and pH, the salt of acquisition is molten
Liquid concentrated combinations again.The process for fixation of the ester hydrolase include but not limited to using existing investment, cross-linking method, covalently
Any one of fixation, absorption method, it is alternative according to the introduction of knowledge and the prior art that those skilled in the art grasp
Ground uses.It is to make hydrolysate (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids of acquisition to the purpose that ester hydrolase immobilizes
Salt impurities are less, and are more advantageous to separation crude product, can improve a crude yield and purity.
For (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters of acquisition, can also be obtained by Base hydrolysis method (S) -
The salt of α-ethyl -2- oxygen -1- pyrrolidine acetic acids：Add 2~3 parts of deionized waters and 2~3 parts of (S)-α-second respectively into reactor
The concentrate of base -2- oxygen -1- pyrrolidine acetic acid ethyl esters, stirring, adding the ionic membrane lye of 1~2 part of 200~400g/L makes instead
Not less than 13,10~20 DEG C 1~5h of hydrolysis of pH are answered, until substrate hydrolysis reaction terminates, obtain (S)-α-ethyl -2- oxygen -1- pyrroles
The salting liquid of alkane acetic acid is coughed up, then the solid crude product of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt is made in concentrated crystallization.
The present invention also provides a kind of cell fixation microbial inoculums, can be by (S)-α-ethyl -2- oxygen -1- pyrroles using the microbial inoculum
Heptane ethyl acetate is splitted out from its racemic modification solution, and the enzyme activity rate of recovery of the cell fixation microbial inoculum is not less than
90%, adsorption rate is not less than 95%, and immobilized microbial inoculum is repeated to be not less than 35 times using number；The microbial inoculum is by the following method
It obtains：
The bacterium solution of methyl packing bacterium cxzy-L013 as described in claim 1 is dissolved in buffer solution, at least adds in a kind of suction
Attached dose and/or a kind of crosslinking agent, stirring filter acquisition immobilized microbial inoculum；The adsorbent be selected from diatomite, activated carbon it is arbitrary
It is a kind of；The crosslinking agent is selected from any one of glutaraldehyde, toluene di-isocyanate(TDI), bis-diazotized benzidine.
Preferably, it carries out cell by the way that absorption method and cross-linking method is used in combination and fixes, the enzyme of immobilised enzymes can be effectively improved
The rate of recovery and adsorption rate living.
Further, after adsorbent is added in, polyethyleneimine is first added in, adds crosslinking agent；The adsorbent choosing
With diatomite, crosslinking agent selects glutaraldehyde.
The producing enzyme somatic cells of Methylopila sp.cxzy-L013 strain fermentations are fixed invention
Change, and utilize the immobilized microbial inoculum to racemic substrate (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters in certain condition
Lower progress stereoselectivity ester hydrolysis reaction, realizes that conversion yield reaches as high as more than 50.0%, and stereoselectivity is good, (S)-α-
Ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl ester enantiomeric excess values e.e.s(%) is not less than 99.5；High catalytic efficiency, resolution reaction
In racemic substrate concentration reach as high as 500g/L, the reaction time is no more than 15 hours, is reused after cell fixation time
Number is easy to industrialized production, downstream separation is simple, and environmental pollution is small not less than 35 times.
Description of the drawings
Fig. 1 is the appearance collection of illustrative plates of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids；
Fig. 2 is the appearance collection of illustrative plates of racemic (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids；
Fig. 3 is the organic phase HPLC figures for being catalyzed resolution of racemic (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters 3h
Spectrum；
Fig. 4 is the water phase HPLC figures for being catalyzed resolution of racemic (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters 3h
Spectrum；
Fig. 5 is the organic phase at the end of catalysis resolution of racemic (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters
HPLC collection of illustrative plates；
Fig. 6 is the water phase HPLC at the end of catalysis resolution of racemic (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters
Collection of illustrative plates；
Fig. 7 is that duration is reacted in test example 3 with the trend chart of enzyme access times.
Specific embodiment
The present invention is further described with reference to specific embodiment, it should be noted that do not carrying out special theory
In the case of bright, percent concentration described below is all mass percent concentration.
Embodiment 1
Methyl packing bacterium cxzy-L013 (Methylopila sp.cxzy-L013), the culture presevation are trained in Chinese Typical Representative
Support object collection (CCTCC), address：Wuhan, China university, 430072, preservation date：On September 18th, 2016, deposit number：
CCTCC NO.：M2016494.
Methyl packing bacterium cxzy-L013 (Methylopila sp.cxzy-L013) is from Linhai city Du Qiao towns Huahai
Through flat-plate bacterial colony feature primary dcreening operation in medicine company on-site soil, using one grade fermemtation shaking flask culture one by one, by detecting enzymatic activity, than
It is obtained compared with stereoselectivity ester hydrolase enzyme activity size.
The feature of the bacterium colony is as follows：Colonial morphology：30 DEG C of 48~72h of culture on LB agar plates, bacterium colony are in regular shape
Circle, neat in edge, 0.5~1mm of diameter, surface elevation, moistening, glossy, milky；Thalline be in short round bar shape, single point
Arrangement is dissipated, size is (0.3~0.4) μ m (1.0~1.2) μm；Gram-negative bacteria；Using glucose, glycerine, ethyl alcohol as carbon
Slow-growing on the culture medium in source, growth is very fast during using methanol, methylamine hydrochloride, ammonium formate as carbon source.
Embodiment 2
For embodiment 1 obtain methyl packing bacterium cxzy-L013, should further activation culture, with carry out fermentation obtain
Obtain the bacterium solution of methyl packing bacterium cxzy-L013.Specific acquisition step is as follows：
Inclined-plane culture
Methylopila sp.cxzy-L013 glycerol tubes bacterial strains are crossed in LB slant tubes, 30 DEG C are cultivated 2~3 days；
Seed liquor culture
Inclined-plane thalline is seeded to seed culture medium：30 DEG C are cultivated 2~3 days, obtain seed liquor；Seed culture medium concentration forms
For：MgSO4·7H2O1.0g/L, K2HPO41.8g/L, (NH4)2SO41.0g/L, yeast extract powder 5.0g/L, volume fraction are
75% methanol solution 5.0mL/L (being added in before inoculation), ammonium hydroxide adjusts pH 7.0.
Inoculation fermentation
Seed liquor is seeded to 7L ferment tanks：Inoculum concentration is 100mL, and zymotic fluid initial volume is 5L, fermentation temperature 30
DEG C, ammonium hydroxide control pH6.5~7.0, ventilatory capacity：0.5vvm, mechanical agitation is gradually increased to 900r/min from 100r/min makes
DO >=30%, the methanol concentration of fermentation process intermittent injecting 75% is 5.0mL/L, ferments 3 days, can be put when pH goes up not down
Tank collects thalline；OD600 >=40 at this time, thalline weight in wet base is up to 70~90g/L.
Fermentation medium concentration forms：NaCl 0.5g/L, MgSO4·7H2O3.6g/L, K2HPO41.0g/L, (NH4)2SO41.0g/L, yeast extract powder 6.0g/L, the methanol solution 5.0mL/L (being added in before inoculation) that volume fraction is 75%.
As shown in table 1, if using the methanol used in fermentation process instead glucose, glycerine, ethyl alcohol as during carbon source, bacterium
Body growth is very slow, it is seen that methanol is significantly better than other three kinds of carbon sources as carbon source.Culture medium prescription used is in addition to carbon source
Remaining is identical with seed liquor culture medium prescription, and cultural method is also identical with seed liquor culture.
Effect of 1 different carbon source of table when bacterium solution is fermented
| Carbon source kind | Carbon source concentration | Fermentation method | Cell concentration OD600 |
| Volume fraction is 75% methanol | 5.0mL/L | Shake flask fermentation | 5±0.5 |
| Glucose | 5g/L | Shake flask fermentation | 2±0.5 |
| Glycerine | 5g/L | Shake flask fermentation | 1±0.5 |
| Volume fraction is 75% ethyl alcohol | 5.0mL/L | Shake flask fermentation | 2±0.5 |
As shown in table 2, it is soaked if using the yeast extract powder in fermentation medium instead Dried Corn Steep Liquor Powder, tryptone, beef
For cream when as carbon source, thalli growth speed is general, and yeast extract powder is better than other three kinds of nitrogen sources.Culture medium prescription used removes
Remaining is identical with seed liquor culture medium prescription outside nitrogen source, and cultural method is also identical with seed liquor culture.
2 different carbon source of table is in the effect as bacterium solution fermentation medium carbon source
The bacteria suspension containing enzyme of 50wt% is no less than to obtain, zymotic fluid is obtained after supercentrifuge centrifuges containing enzyme
Wet thallus is stirred evenly wet thallus and water dilution by equal quality ratio, and refrigeration is for use；Or directly by zymotic fluid through micro-filtration membrane concentration extremely
Mass fraction containing wet thallus is about 50% bacteria suspension, and refrigeration is for use.
Embodiment 3
(S)-α-ethyl -2- oxygen -1- pyrroles are prepared using 1 methyl packing bacterium cxzy-L013 stereoselectivity fractionations of embodiment
Alkane acetate is coughed up, is included the following steps：
(1) bacterium solution of methyl packing bacterium cxzy-L013 is handled by method for immobilizing cell, obtains immobilized microbial inoculum；First
The bacterium solution acquisition pattern of base packing bacterium cxzy-L013 has illustrated in example 2.
(2) a certain amount of water and immobilization are added in for substrate with (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester
Microbial inoculum carries out resolution reaction, obtains (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters；
(3) (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt is obtained using enzyme hydrolysis or basic hydrolysis.
(4) (R)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in water phase are recycled after step (2), through racemization and into ester
Starting material raw material is obtained after change.
Wherein, step (1) method for immobilizing cell is first to add in adsorbent, and crosslinking agent is added after being uniformly mixed.
Embedding bead is formed, then combine by cross-linking method first with absorption method, mechanical strength can be significantly increased.Method for immobilizing cell is real
The enzyme activity rate of recovery of existing immobilised enzymes is not less than 90%, and adsorption rate is not less than 95%, and immobilized microbial inoculum, which repeats, utilizes number
Not less than 35 times.
In step (2), (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester is the toluene solution of 500~900g/L,
Preferred concentration is 700~800g/L.(R, the S)-α-quality of ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester in the reaction system is dense
It spends for 160~500g/L.Immobilized microbial inoculum is in terms of weight in wet base, a concentration of 5~25g/L of throwing amount in the reaction system.
The condition of resolution reaction：Reaction temperature is 25~37 DEG C, and the sodium carbonate (V/V) that volume fraction is 15~25% is added dropwise
Aqueous solution control reaction process pH 6.5~8.5, reacts 2~15h, and reaction conversion results, enantiomeric excess are detected using HPLC
Value e.e.s(%) not less than 99.5, conversion ratio is up to 50%.
Mixed liquor isolation and purification method after resolution reaction is：By reaction solution directly with supercentrifuge centrifugation or board-like mistake
Filter filters, and retains organic phase after liquid portion layering, and the toluene that 1: 0.3~1.5 are added in water layer carries out 3~5 extractions, closes
And organic phase, 35~60 DEG C steam toluene, obtain concentrate (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters.
Any ester hydrolase that can hydrolyze (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl ester ester bonds all may be used in step (3)
To use.Add 1 part of industrial water and 1~2 part of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters respectively into reactor
Concentrate, add in 1/20~1/5 part of ester hydrolase, stirring controls temperature and pH, until ester hydrolysis reaction terminates, filters out ester
Hydrolase obtains the salting liquid of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids, then (S)-α-ethyl -2- is made in concentrated crystallization
The solid crude product of oxygen -1- pyrrolidine acetic acid salt.
It, can also be by basic hydrolysis side for (S)-α of acquisition-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters in step (3)
Method obtains the salt of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids：Add 2~3 parts of deionized waters and 2~3 respectively into reactor
The concentrate of part (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters, is stirred, and adds the ionic membrane caustic soda of 1~2 part of 300g/L
Liquid makes pH reach 14,10~20 DEG C of 1~5h of hydrolysis, until substrate hydrolysis reaction terminates, obtains (S)-α-ethyl -2- oxygen -1- pyrroles
The salting liquid of alkane acetic acid is coughed up, then the solid crude product of (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt is made in concentrated crystallization.
Embodiment 4
The step of the present embodiment has advanced optimized method for immobilizing cell in step (1) based on embodiment 3：To contain wet bacterium
It is preference that the bacteria suspension 20g that weight score is 50%, which is dissolved in 100mL Ammonium formate buffers (pH 7.0) to carry out reaction, is stirred
It mixes uniformly mixed, adds 0.4~0.8g diatomite or activated carbon, 5% 3~4.5mL of polyethyleneimine is added to be crosslinked 1h, then add 25% penta
1~1.5mL of dialdehyde is crosslinked 1h, and finally vacuum filtration obtains immobilized microbial inoculum, with originally water washing 2 times and filters, be placed in 4 DEG C it is cold
It hides and preserves.The enzyme activity rate of recovery >=90% of immobilised enzymes, adsorption rate >=95%.
Embodiment 5
Method of the present embodiment based on embodiment 3 selects the Protin AP Conc. fat of Japanese amano enzyme preparation company
Enzyme obtains (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt as ester hydrolase.
Above-mentioned ester hydrolase powder 2g is dissolved in 100mL purified waters, adds in sodium alginate 2g, is slowly stirred until complete
Mixed liquor syringe needle or nozzle are uniformly pumped into the calcium chloride solution of 100mM, and are slowly stirred by fully dissolved, treat pearl gel
It is washed repeatedly with distilled water 2~3 times after hardening, obtains calcium-alginate-immobilized enzyme, be placed in 4 DEG C and preserve for use.
Embodiment 6
A kind of cell fixation microbial inoculum, using the microbial inoculum can by (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester from its
It is splitted out in racemic modification solution, the enzyme activity rate of recovery of immobilised enzymes is not less than 90%, and adsorption rate is not less than 95%, immobilization
Microbial inoculum is repeated to be not less than 35 times using number；It obtains by the following method：
The bacterium solution of methyl packing bacterium cxzy-L013 (Methylopila sp.cxzy-L013) described in embodiment 2 is molten
In buffer solution, diatomite is at least added in as adsorbent, polyethyleneimine is then added in, adds glutaraldehyde as crosslinking
Agent, stirring to obtain immobilized cell solution.Vacuum filtration obtains the immobilized microbial inoculum.Specific method is with reference to described in embodiment 4.
Embodiment 7
The present embodiment further describes (S)-α in step (2)-ethyl -2- oxygen -1- pyrrolidine acetic acid second based on embodiment 3
The step of ester is split：
By taking 300mL catalystic converter systems as an example：(R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl ester concentration is about
The toluene solution 100mL of 500g/L prepares cell fixation microbial inoculum according to the method for embodiment 4 and embodiment 6.
Specially：Add water 200mL in the conversion bottle of 500mL, add in (R, S)-α-ethyl -2- oxygen -1- pyrroles containing about 50g
Heptane ethyl acetate toluene solution 100mL is coughed up, makes (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester in the reaction system
Initial concentration is about 166g/L, opens stirring, uses Na2CO3PH is adjusted to 7.0 by solution (20%, V/V), is added in embodiment 6 and is prepared
Cell fixation microbial inoculum 2g makes immobilized microbial inoculum a concentration of 6.6g/L of throwing amount, 37 DEG C, uses Na2CO3Solution (20%, V/V) is by pH
7.0~7.5 are maintained, 2~3h of resolution reaction.Reaction conversion results are detected using HPLC, enantiomeric excess value isConversion ratio is 50.0%.
8~embodiment of embodiment 13
The method for splitting of 8~embodiment of embodiment 13 is substantially the same manner as Example 7, but the throwing amount of cell fixation microbial inoculum,
Concentration, substrate concentration in the reaction system of the substrate used in toluene solution are different, as can be seen from Table 3 different embodiments
Split the difference of effect.
Table 3 biocatalytic resolution substrate (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl ester results
It is understood based on 8~embodiment of embodiment 13：When the toluene solution throwing amount of substrate is more, the reaction time is longer, turns
Rate is declined；Concentration of the substrate in toluene solution is higher, and throwing amount in the reaction system is bigger, and reaction can be prolonged
It is long, it can make conversion ratio that can still reach ideal effect by suitably increasing the method for immobilized microbial inoculum throwing amount.
1 resolution reaction liquid of test example monitors
Instrument：High performance liquid chromatograph is equipped with UV detector
Chromatographic column：CHIRALPAK AS-H 250×4.6mm 5μm
Mobile phase：N-hexane: isopropanol: trifluoroacetic acid=80: 20: 0.2 (%V/V/V)
Flow velocity：0.8mL/min, wavelength：210nm, column temperature：30 DEG C, run time：30min, sample size：20μL
Dilution：Mobile phase, blank solution：Dilution
Test solution：It weighs 20mg test samples to be placed in 10mL volumetric flasks, be dissolved with dilution, constant volume.Test sample packet
It includes：Racemic (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester and monomer, racemic (R, S)-α-ethyl -2- oxygen -
1- pyrrolidine acetic acids and monomer.
Sample treatment：Based on the method and step of embodiment 4,100 times of dilution is extracted reaction solution, with 0.45 μm of micropore after mixing
Membrane filtration treats sample introduction.
(S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester enantiomeric excess value e.e. and substrate conversion efficiency C presses following public affairs
Formula calculates：
In formula, [C]S[C]RRespectively chromatography measures the content of substrate S and R type in sample, e.e.s(%) is anti-to split
The enantiomeric excess value of (the S)-α answered-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters, CPFor the molar concentration of product, CSFor residue
Substrate molar concentration, C (%) are conversion ratio.
(S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters appearance time be can be seen that from Fig. 1 to Fig. 6 as 9.4min,
(R)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters appearance time is 15.9min, (S)-α-ethyl -2- oxygen -1- pyrrolidines second
Sour appearance time is 11.5min, and (R)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids appearance time is 10.4min.
Fig. 5 is that the HPLC at the end of catalysis resolution of racemic (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters is organic
Phase collection of illustrative plates, it can be seen that (R)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters have hydrolyzed completely.
The comparison of 2 process for fixation of test example
The present invention, simultaneously using adsorbent and crosslinking agent, is improved in process for fixation with polyethyleneimine cooperation glutaraldehyde
Cross-linking effect, the fixed effect of cell are obviously improved, so again with high concentration organic substrates participate in enzymic catalytic reaction when,
It is easily industrialized production purpose.
Immobilization object：Somatic cells after Methylopila sp.cxzy-L013 strain fermentations and bacterium in embodiment 2
The enzyme solution that body cell is obtained through ultrasonic disruption.
First, use of this test example respectively with regard to absorption method, investment and absorption-cross-linking method is compared, and explain
The difference of the fixed effect of enzyme in the cell caused by bacterium solution processing method difference is stated, the results are shown in Table 4：
The influence of table 4 different fixing means and bacterium solution processing method to fixed effect
Secondly, for test group D, difference again consolidates adsorbent (diatomite or activated carbon) and the dosage of crosslinking agent
Surely change Optimal Experimental, the results are shown in Table 5：
The influence of table 5 different fixing means and bacterium solution processing method to fixed effect
3 cell fixation of test example reuses number verification
Verification method：Immobilized microbial inoculum catalysis 100mL racemics (R, the S)-α-ethyl-2- oxygen obtained with 5g embodiments 4-
1- pyrrolidine acetic acid ethyl esters, content of the racemic substrate in toluene is 500g/L, reaction system 300mL, with consumption
30mLNa2CO3Solution (20%, V/V) is reaction end, and immobilized microbial inoculum after reaction is filtered, will be filtered
Immobilized microbial inoculum puts into same system, and carries out next secondary response in same reaction conditions.
Fig. 7 is to react the trend chart for terminating required time as access times increase, as can be seen from the figure the 1st time
Reaction terminates to need 1.2h, hereafter reacts duration and gradually increases, and the 7th time is 2.6h, but the 8th secondary response duration starts to reduce again,
In view of since enzyme repeats to filter and can be lossy using weight, W-response takes in rising trend after the 12nd time, but thin
Reaction duration is maintained essentially in 5h after born of the same parents' immobilized microbial inoculum has used 36 times, and effect is preferable, reaches expected.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (16)
1. a kind of methyl packing bacterium (Methylopila sp.) cxzy-L013 bacterial strains, in September in 2016 is preserved on the 18th
State's Type Tissue Collection, deposit number are CCTCC NO：M2016494.
2. methyl packing bacterium as described in claim 1 prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation
Application on salt.
3. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation according to claim 2
Application on salt, it is characterised in that including following 3 step：
(1) bacterium solution of methyl packing bacterium is handled by method for immobilizing cell, obtains immobilized microbial inoculum；
(2) a certain amount of water and the immobilization are added in for substrate with (R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids ethyl ester
Microbial inoculum carries out resolution reaction, obtains (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters；
(3) and then using enzyme hydrolysis or basic hydrolysis (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt is obtained.
4. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation according to claim 3
Application on salt, it is characterised in that：The bacterium solution of the methyl packing bacterium is to train methyl packing bacterium through inclined-plane culture, seed liquor
It supports, the bacteria suspension containing enzyme that 50wt% is no less than containing wet thallus that inoculation fermentation step obtains.
5. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation according to claim 3
Application on salt, it is characterised in that：The method for immobilizing cell is that the bacterium solution of methyl packing bacterium is dissolved in buffer solution, at least
A kind of adsorbent and/or a kind of crosslinking agent are added in, stirring filters acquisition immobilized microbial inoculum；
The adsorbent is selected from any one of diatomite, activated carbon；
The crosslinking agent is selected from any one of glutaraldehyde, toluene di-isocyanate(TDI), bis-diazotized benzidine.
6. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation according to claim 5
Application on salt, it is characterised in that：The enzyme activity rate of recovery for the immobilised enzymes that the method for immobilizing cell is realized is not less than 90%,
Adsorption rate is not less than 95%, and immobilized microbial inoculum is repeated to be not less than 35 times using number.
7. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation according to claim 6
Application on salt, it is characterised in that：The method for immobilizing cell is first to add in adsorbent, and friendship is added after being uniformly mixed
Join agent.
8. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation according to claim 7
Application on salt, it is characterised in that：Polyethyleneimine is also added into when adding in the crosslinking agent.
9. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acids in selective fractionation according to claim 3
Application on salt, it is characterised in that：The temperature of the resolution reaction is 25~37 DEG C, and the sodium carbonate of 15~25v/v% is added dropwise
Solution control reaction process pH 6.5~8.5, reacts 2~15 hours.
10. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidines second in selective fractionation according to claim 9
Application on hydrochlorate, it is characterised in that：(R, S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid ethyl esters are anti-in the resolution reaction
It is 160~500g/L to answer the mass concentration in system.
11. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidines second in selective fractionation according to claim 10
Application on hydrochlorate, it is characterised in that：The immobilized microbial inoculum in terms of weight in wet base, throwing amount a concentration of 5 in the reaction system~
25g/L。
12. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidines second in selective fractionation according to claim 11
Application on hydrochlorate, it is characterised in that：HPLC detection enantiomeric excess values e.e. is utilized after resolution reactions(%) is not less than
99.5, conversion ratio is not less than 49%.
13. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidines second in selective fractionation according to claim 3
Application on hydrochlorate, it is characterised in that：(R)-α-ethyl -2- oxygen -1- pyrrolidines second in water phase is recycled after the step (2)
Acid, through racemization and into obtaining starting material raw material after esterification.
14. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidines second in selective fractionation according to claim 3
Application on hydrochlorate, it is characterised in that：The step (3) by immobilized ester hydrolases obtain (S)-α-ethyl -2- oxygen -
1- pyrrolidine acetic acid salt, then concentrated crystallization obtain its crude product.
15. methyl packing bacterium prepares (S)-α-ethyl -2- oxygen -1- pyrrolidines second in selective fractionation according to claim 3
Application on hydrochlorate, it is characterised in that：Lye used in step (3) basic hydrolysis is the ionic membrane caustic soda of 200~400g/L
Liquid makes pH value in reaction not less than 13,10~20 DEG C of reaction temperature, until substrate hydrolysis reaction terminates, then concentrated crystallization obtains
Its crude product.
16. a kind of cell fixation microbial inoculum, it is characterised in that：It can be by (S)-α-ethyl -2- oxygen -1- pyrrolidines second using the microbial inoculum
Acetoacetic ester is splitted out from its racemic modification solution, and the enzyme activity rate of recovery of the cell fixation microbial inoculum is inhaled not less than 90%
Attached rate is not less than 95%, and immobilized microbial inoculum is repeated to be not less than 35 times using number；The microbial inoculum obtains by the following method：
The bacterium solution of methyl packing bacterium as described in claim 1 is dissolved in buffer solution, at least adds in a kind of adsorbent and/or one kind
Crosslinking agent, stirring filter acquisition immobilized microbial inoculum；The adsorbent is selected from any one of diatomite, activated carbon；The crosslinking
Agent is selected from any one of glutaraldehyde, toluene di-isocyanate(TDI), bis-diazotized benzidine.
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| CN201611106084.2A CN106591179B (en) | 2016-12-05 | 2016-12-05 | Methyl packing bacterium and its prepare the application on (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt in selective fractionation |
| JP2019549623A JP6720420B2 (en) | 2016-12-05 | 2017-09-19 | Use in the preparation of (S)-α-ethyl-2-oxo-1-pyrrolidine acetate by methylopira and its selective resolution |
| EP17877976.5A EP3533862B1 (en) | 2016-12-05 | 2017-09-19 | Methylopila sp. and use thereof in selective resolution and preparation of (s)-alpha-ethyl-2-oxo-1 -pyrrolidine acetate |
| BR112019011554A BR112019011554A2 (en) | 2016-12-05 | 2017-09-19 | methylopyl sp. and use thereof in the preparation of selective resolution of (s) -alpha-ethyl-2-oxo-1-pyrrolidine acetate |
| US16/466,171 US11085059B2 (en) | 2016-12-05 | 2017-09-19 | Methylopila sp. and use thereof in selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate |
| PCT/CN2017/102275 WO2018103409A1 (en) | 2016-12-05 | 2017-09-19 | METHYLOCYSTIS AND USE THEREOF IN SELECTIVE RESOLUTION AND PREPARATION OF (S)-α-ETHYL-2-OXO-1-PYRROLIDINE ACETATE |
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| WO2019028671A1 (en) * | 2017-08-08 | 2019-02-14 | 浙江华海药业股份有限公司 | Method for preparing levetiracetam |
| WO2019028666A1 (en) * | 2017-08-08 | 2019-02-14 | 浙江华海药业股份有限公司 | Method for synthesizing levetiracetam |
| CN109234187B (en) * | 2018-08-15 | 2021-06-15 | 李晓明 | Atrazine degrading bacterium and application thereof |
| CN113816872A (en) * | 2020-06-19 | 2021-12-21 | 浙江华海药业股份有限公司 | Synthesis method of (S) -2-aminobutanamide |
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| BR112019011554A2 (en) | 2019-10-22 |
| EP3533862B1 (en) | 2021-04-28 |
| JP6720420B2 (en) | 2020-07-08 |
| WO2018103409A1 (en) | 2018-06-14 |
| CN106591179A (en) | 2017-04-26 |
| US11085059B2 (en) | 2021-08-10 |
| EP3533862A1 (en) | 2019-09-04 |
| EP3533862A4 (en) | 2019-09-04 |
| US20190360011A1 (en) | 2019-11-28 |
| JP2020500555A (en) | 2020-01-16 |
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